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Protein stability detection method and application

A technology for labeling proteins and target proteins, applied in the field of protein detection, can solve the problems of limited recognition ability of mass spectrometry technology, limited technical practicability, undetectable changes in the stability of low-abundance proteins, etc.

Active Publication Date: 2014-10-15
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Protein microarray technology can detect the stability of multiple proteins at one time, but due to the availability of antibodies, its throughput is limited, which also greatly limits the practicality of this technology
Mass spectrometry can be used to detect large-scale samples and protein stability changes within the scope of proteomics, but mass spectrometry has limited recognition capabilities and cannot detect changes in the stability of low-abundance proteins

Method used

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  • Protein stability detection method and application
  • Protein stability detection method and application
  • Protein stability detection method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0211] Example 1. The dual fluorescent protein system efficiently measures the relative stability of proteins

[0212] The inventor designed and constructed a dual fluorescent protein structure, in which mEGFP is fused to the C-terminus of the target protein, the intermediate linker protein is ubiquitin with K(R) saturation mutation, and the internal reference fluorescent protein is mRFP, and the three constitute mEGFP fu -mRFP f The structure, wherein mEGFP reference sequence SEQ ID NO:1,2, mRFP reference sequence SEQ ID NO:3,4, Ub K0 The reference sequence is SEQ ID NO: 17, 18, and its structural diagram is as follows figure 1 As shown in a, the principle of relative quantification of the target protein is as follows figure 1 As shown in b, the inventors constitutively expressed the artificially designed recombinant dual fluorescent protein structural components on mammalian cell expression plasmids using CMV promoters.

[0213] The inventors will not be fused with any fo...

Embodiment 2

[0217] Example 2. mEGFP-UB K0 -mRFP dual fluorescent protein system detects protein stability across the genome

[0218] The inventor used human ORFeome V5.1 as the target gene library, and used gateway homologous recombination technology to fuse the C-terminals of nearly 15,000 ORFs to mEGFP-UB K0 - mRFP structure. Using fusion with mEGFP fu -mRFP f The gene library of the structure was used to infect 293 cells with lentivirus infection technology, and a dual fluorescent cell line integrated with the library gene was constructed. see results Figure 6 . Then, using FACS technology, based on the ratio of mEGFP / mRFP, the library was sorted into 8 intervals, and the genomes of cells in each interval were extracted, and PCR products were used as templates for in vitro cRNA amplification, followed by chip hybridization. The Composite loess normalization program performs data processing and calculates the relative stability parameter PSI (Protein Stability Index) of each gene...

Embodiment 3

[0219] Example 3. Detection of candidate genes responding to the anticancer drug bortezomib at the level of protein stability using a dual fluorescent cell library

[0220] The inventors used the double fluorescent cell library containing the human ORFeome V5.1 gene library, treated the library cells with DMSO (control treatment) and Bortezomib (experimental treatment) respectively, and then used figure 2 The process shown in a, respectively calculate the PSI value of each library gene in the state of DMSO and Bortezomib, and then calculate △PSI=PSI BTZ -PSI DMSO , and then use △PSI to rank all genes, and conduct bioinformatics analysis on significant genes. image 3 a is the chemical structure of the anticancer drug Bortezomib, image 3 c, d are representative data with substrate-responsive proteins, Figure 4 The results were analyzed for bioinformatics.

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Abstract

The invention provides a genetic constructing matter with a structure shown as 5'-A + B + C + D + E-3', A represents a promoter; B represents a coding sequence of a fusion protein comprising a target protein and a first marker protein; C represents a connecting peptide coding sequence; D represents a coding sequence of a second marker protein; and E represents a terminator. The invention also provides polypeptides with a structure shown as C1 + B1+ D1, B1 represents a fusion protein composed of the target protein and the first marker protein; C1 represents a connecting peptide; and D1 represents a coding sequence of a second marker protein. The invention also provides a carrier comprising the genetic constructing matter, mammalian cells containing the genetic constructing matter or vectors, a library formed by the cells, a protein stability detection method and application. The protein stability detection method is high in sensitivity and specificity in protein stability detection and is easy to operate.

Description

technical field [0001] The present invention relates to the field of protein detection. In particular, the present invention relates to methods and applications for detecting protein stability. Background technique [0002] The dynamic change of protein level is the basic feature and necessary prerequisite for cells to maintain vitality and carry out life activities. At any point in time, the level of a particular protein within a cell depends on the dynamic balance between its synthesis and degradation processes. Protein synthesis is mainly carried out on ribosomes, and the process of protein synthesis and its regulation is relatively clear. [0003] Human research on protein degradation process began in the 1980s, and has developed into a very important research field of modern biology in recent years. This is because all proteins must be degraded in a timely manner so that the basic life processes of cells such as the cell cycle can be made possible. Numerous evidence...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00C12N15/867C12N5/10C40B40/02G01N33/68C12Q1/68
CPCC12N15/62G01N21/64C12N15/65C07K2/00C07K2319/00C07K14/47C07K2319/95C07K14/435C07K2319/60C12N15/85
Inventor 胡荣贵于涛陶永辉
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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